Pos0332 identification of Distinct Cellular Populations in the Synovium of Early- And Late-Stage Radiographic Knee Oa Using Single Nucleus Rna Sequencing

Abstract

BackgroundOsteoarthritis (OA) is a complex, multifactorial and heterogeneous joint disease of unknown etiology. OA research has largely focused on articular cartilage degeneration with little attention given to other joint tissues, including the synovium. The synovium lines the joint capsule, produces synovial fluid for lubrication, and is emerging as a contributor to OA pathogenesis. In OA, the synovium exhibits increased vascularization, inflammation, hyperplasia and fibrosis. Synovial cells that contribute to these pathological events during early and advanced stages of OA are not well characterized. The emergence of RNA sequencing (RNAseq) at the resolution of a single cell or nucleus allows for the identification of distinct cells that may contribute to OA pathogenesis.ObjectivesTo delineate the synovium’s role in OA pathogenesis we sought to identify if distinct cell subtypes exist in the synovium of early (KL1) versus late stages (KL3/4) of radiographic knee OA using single nucleus RNA sequencing.MethodsSynovia from patients with early (KL=I; n=5) and late (KL =III/IV; n=4) stage radiographic knee OA were subjected to single nucleus (sn)RNAseq and to bioinformatics analyses. Canonical cell-specific markers were used to identify cell types from the unsupervised clustering analysis and prominent cell types were re-clustered. Differentially expressed gene (DEG) lists between the subclusters were determined based on top gene expression within a cell type between early and late OA synovium. Cell surface markers identified from the DEGs were validated by immunohistochemistry. Pathway and gene ontology enrichment analysis were performed on fibroblast subclusters to identify prominent pathways and transcription factors that were upstream regulators. Ongoing in vivo and in vitro methods are being used to assess these transcription factors in both fibroblast cell culture and an OA mouse model.ResultsFibroblasts and macrophages constituted 75% of the cells from early and late-stage synovium and re-clustering analysis resolved 8 fibroblast and 6 transcriptionally distinct macrophage subclusters (Figure 1). Cluster based nuclei proportion differences identified fibroblast clusters 1, 2, 4 and 6 and macrophage clusters 1, 2 and 5 to contribute to early-stage samples while fibroblast clusters 0, 3 and 5 and macrophage clusters 0, 3 and 4 to late-stages. Downstream analyses focused on fibroblasts and putative cell surface markers from fibroblast subclusters were identified from DEGs and confirmed by immunohistochemistry. The fibroblast subclusters were subjected to pathway analyses which identified clusters 0 and 1 to be the most prominent clusters which both shared common ECM related pathways. Upstream transcription factors that regulate ECM related genes were identified for both subclusters 0 and 1. Current efforts are focussed on selecting and targeting transcription factor(s) for both in vitro and in vivo analyses by utilizing siRNA’s in fibroblast culture and a cre-lox mouse system to identify the mechanisms associated with the synovial pathology during OA.ConclusionSnRNAseq analysis identified distinct subclusters of fibroblasts and macrophages to exist in human OA knee synovia. Certain subclusters were more representative of the early stage while others were more representative of the late stage of the disease. Further validation studies are being performed to assess the functional roles of these subclusters and whether targeting them would attenuate disease progression.KT and EG share equal first author contribution.DE and Mk share equal senior author contribution.AcknowledgementsCanadian Institute of Health Research.Disclosure of InterestsNone Declared.